DP095 | INTERACTION BETWEEN APOPTOSIS, PHOSPHOINOSITIDE-DEPENDENT PATHWAYS, AND POTENTIAL IMPLICATIONS OF A NOVEL BCL2 MUTATION IN RESPONSE TO AZACITIDINE AND VENETOCLAX IN HIGH-RISK MYELODYSPLASTIC NEOPLASMS

Azacitidine (AZA) is a standard hypomethylating agent for high-risk Myelodysplastic Neoplasms (MDS). The addition of Venetoclax (VEN), a BCL-2 inhibitor, enhances its efficacy. This study investigates the molecular effects of AZA and AZA+VEN on apoptotic and proliferative pathways in high-risk MDS patients and leukemic cell lines. Nine high-risk MDS patients (IPSS-R: intermediate to very high) treated at the IRCCS Institute of Hematology "L. e A.Seràgnoli," Bologna, received either AZA alone (n=5) or AZA+VEN (n=4). Baseline bone marrow or peripheral blood samples were analyzed for gene mutations, and qPCR analyses were performed during treatment. AZA responders (R) —achieving Complete Remission (CR) or Hematological Improvement (HI)— showed reduced BCL2 and increased BAX in early cycles, while non-responders (NR) — with Stable Disease (SD) — showed the opposite trend. All AZA+VEN-treated patients responded (CR or HI), with an initial increase followed by downregulation of both BCL2 and BAX. Notably, in one AZA+VEN-treated patient with CR and later progression to AML, BCL2 levels rose shortly before evolution, while BAX remained stable. Additionally, a recurrent BCL2 frameshift mutation (p.Ala82Glyfs*14) was identified in 7/9 patients at baseline, potentially generating a truncated protein. While not directly linked to treatment response overall, all AZA+VEN treated patients harboring this mutation exhibited a 100% response rate, compared to a 25% among mutated patients treated with AZA alone. At baseline, mutated AZA-NR exhibited higher expression levels of BCL2, CD33, CD11b, and PLCG2 compared to mutated AZA+VEN-R. To further characterize the molecular pathways underlying AZA+VEN, we analyzed VEN-sensitive MV4-11 and VEN-resistant THP-1 cells treated with AZA, VEN, or AZA+VEN for 24 hours. Particularly in MV4-11 cells, AZA+VEN triggered a pro-apoptotic response, with increased Annexin V staining, caspase activity, PARP cleavage, and reduced progression in G0-G1 and G2-M. We also observed BCL2 and PLCG2 protein levels downregulation, reduced Akt phosphorylation, and increased GSK3B dephosphorylation. All in all, our data indicate a possible crosstalk between the phosphoinositide and apoptotic pathways in AZA+VEN’s mechanism of action. The BCL2 mutation may enhance sensitivity to AZA+VEN by modulating these pathways. Ongoing studies are now focusing on expand the patient cohort, generate BCL2-mutant lines, and explore mechanisms in AZA+VEN-resistant models.